PROJECT SUMMARY Lung cancer is the leading cause of cancer death in the United States. The development of new and effective chemopreventive agents for lung cancer is urgently needed. Our long-term goal is to identify and advance new and efficacious preventive agents targeting human lung cancer. There is growing evidence that distinct tumor- specific metabolic changes, including reliance on aerobic glycolysis and changes in mitochondrial bioenergetics, are key drivers of malignancy. We made chemical modifications to lonidamine (LON), an anti-glycolytic compound with limited anti-tumor efficacy, to create Mito-LON as a more effective and safe mitochondria- targeted, tumor cell selective agent with a new mechanism, specifically OXPHOS inhibition. Our preliminary data show that Mito-LON, at low micromolar concentrations, is a potent inhibitor of cancer cell mitochondrial bioenergetics, and results in pronounced mitigation of lung cancer development, cell proliferation, growth, progression, and metastasis. We hypothesize that Mito-LON inhibits lung tumor development and metastasis through induction of autophagic cell death (ACD) by suppressing mitochondrial complexes I and II, depleting cellular ATP, stimulating ROS formation, and subsequent effects on AKT/mTOR/p70S6K signaling. We will test our hypothesis in three specific aims. Aim 1 will determine the effects of Mito-LON on mitochondrial bioenergetics and redox status in cellular systems in vitro. Aim 2 will determine the capacity of Mito-LON to induce ACD, with a focus on mitophagy, as a mechanism to mitigate lung cancer progression and metastasis. Aim 3 will determine the capacity of Mito-LON to inhibit lung tumor progression and lung cancer brain metastasis in vivo. We will use state-of-the-art small animal imaging to monitor the growth of primary tumors (using magnetic resonance imaging) and brain metastasis (through bioluminescence imaging). Successful completion of these aims will increase our understanding of the molecular basis of autophagy in the context of lung cancer inhibition and more broadly establish a new approach for using mitochondria-targeting drugs to effectively and selectively block cancer cell metabolism, energy generation, and induce ACD. This proposal is timely and significant since future clinical trials of Mito-LON against lung cancer will require vigorous preclinical characterization of the efficacy and precise mechanisms of action in targeting cancer metabolism.